In this study, melatonin (MT) promoted lipid accumulation in Monoraphidium sp. QLY-1 under nitrogen deficiency conditions. The lipid accumulation increased 1.22- and 1.36-fold compared with a nitrogen-starved medium and a normal BG-11 medium, respectively. The maximum lipid content was 51.38%. The reactive oxygen species (ROS) level in the presence of melatonin was lower than that in the control group, likely because of the high antioxidant activities. The application of melatonin upregulated the gibberellin acid (GA) production and rbcL and accD expression levels but downregulated the abscisic acid (ABA) content and pepc expression levels. These findings demonstrated that exogenous melatonin could further improve the lipid production in Monoraphidium sp. QLY-1 by regulating antioxidant systems, signalling molecules, and lipid biosynthesis-related gene expression under nitrogen deficiency conditions.